This invention relates to a novel method of determining vitamin D metabolites.
It is well known that vitamin D plays an active role in the homeostatic mechanisms that control calcium metabolism. That is, this vitamin is involved in the precise control of the concentration of the calcium ion in plasma. Vitamin D is transported to various sites in the body where it is activated. The activated forms of the vitamin act on the target tissues, thereby causing an increase in calcium content. The activation of vitamin D is regulated in a negative feedback system by plasma calcium.
The most biologically active form of vitamin D is 1,25-dihydroxycholecalciferol or calcitriol, 1,25-(OH).sub.2 D.sub.3, which is formed by two successive hydroxylations of vitamin D.sub.3. That is, calcitriol is formed by the sequential hydroxylation of vitamin D.sub.3 at C-25 in the liver and at C-1 in the kidney. Various other analogs can be produced by hydroxylation at C-24 and C-26. Vitamin D.sub.3, cholecalciferol, is produced in the body when the skin which contains the provitamin 7-dehydrocholesterol is exposed to sunlight.
Calcitriol functions primarily in intestinal calcium transport and bone calcium resorption. Abnormalities in the metabolism of calcitriol, as manifested by circulating levels of the compound, have been shown to play roles in the pathogenesis of a variety of diseases such as renal osteodystrophy, sarcoidosis and post-menopausal osteoporosis, a disorder which is endemic in western society.
Accordingly, the determination of circulating levels of calcitriol is of significant interest in the medical field. The assay of calcitriol in human blood serves as an excellent index of the status of vitamin D metabolism and provides a useful adjunct to other methods of determining vitamin D deficiency states.
Development of assay methods for calcitriol have been difficult because of its extremely low concentration in blood. Background information on these difficulties can be had by reference, for example, to Haussler et al., New England J. of Med. 297(19), 1041-50 (1977).
The principal assay for calcitriol in serum which has been developed heretofore is a radioreceptor assay, or competitive protein binding assay. This assay employs an ardously obtained, unstable rachitic chick intestinal binding protein. Moreover, due to the relative lack of specificity of this cytosolic receptor, each sample requires extensive chromatographic extraction and purification before it can be assayed. This standard assay which employs high pressure liquid chromatography (HPLC) is illustrated, for example, by
Dokoh et al., Anal. Biochem. 116, 211-222 (1981); PA0 Eisman et al., Arch. Biochem. Biophys. 176, 235-243 (1976); and PA0 Gray et al., J. Clin. Endocrin. and Metab. 45, 299-306 (1977).
Because of these difficulties, the standard radioreceptor assay is performed only in a relatively few clinical laboratories. In an effort to procure more stable and specific reagents, polyclonal antibodies to vitamin D metabolites have been produced and used as high affinity binding agents in radioimmunoassays for these compounds. See, for example, Clemens et al., Clin. Endocrinol. 11, 225 (1979). Monoclonal antibodies share the stability of polyclonal antibodies, but can be readily screened for a particular specificity that will discriminate among a large number of closely related compounds as reported by Coffins et al., J. Cell Physiol. 79, 429 (1972). Since such clones could be theoretically maintained indefinitely, their development could circumvent present difficulties of the standard calcitriol serum assay. Previously, we have reported on a radioreceptor assay for calcitriol which employs a highly sensitive monoclonal antibody for the binding protein. Perry et al., International Workshop on Vitamin D, Williamsburg, Va., February 1982, Abstracts, p. 70. However, such assay still requires the use of high pressure liquid chromatography as an essential part of the assay. Said assay employs calcitriol bound to the carrier protein bovine serum albumin as the immunogen for preparing the monoclonal antibody.